Role of p85 subunit of phosphatidylinositol-3-kinase as an adaptor molecule linking the insulin receptor to insulin receptor substrate 1.

Abstract

After insulin stimulation of cells, signaling complexes are formed, containing the insulin receptor (IR), insulin receptor substrate-1 (IRS-1), and phosphatidylinositol-3-kinase. To study the nature of these complexes, we employed purified IR, recombinant IRS-1, antibodies to IR and IRS-1, and fusion proteins containing the two SH2 domains of p85. In intact cells, insulin increased tyrosine phosphorylation of both the IR and IRS-1. Both of these proteins were immunoprecipitated with antibodies to p85. Also, fusion proteins containing the two SH2 domains of p85 directly precipitated both the IR and IRS-1. Next, these signaling complexes were reconstituted in vitro with purified IR, recombinant IRS-1, and the two SH2 domains of p85. In the presence of both SH2 domains of p85, the IR associated with IRS-1. Other data, both in intact cells and in vitro, demonstrated that N- and C-terminal SH2 domains of p85 had preferential binding affinities for the IR and IRS-1, respectively. Studies with an IR mutant truncated in the C terminus indicated that the C-terminal phosphotyrosines of the IR play a major role in interacting with the SH2 domains of p85. In conclusion, both in vivo and in vitro data support a role for p85 in directly linking the IR to IRS-1 via its SH2 domains. The formation of these complexes, therefore, may provide a mechanism for the translocation to the plasma membrane of phosphatidylinositol-3-kinase and other molecules that are involved in IR signaling.